The small GTP binding protein superfamily (consisting of small, monomeric GTP hydrolyzing proteins or GTPases) regulates diverse cellular processes including gene expression, cellular proliferation, cytoskeletal reorganization, vesicle trafficking, and nucleocytoplasmic transport. Activated point mutants of Ras GTPases have been found in over 30% of all human tumors, demonstrating a key role for these proteins in carcinogenesis. The Rap GTPases, which share about 50% sequence identity with Ras, have also been shown to promote activation of MAP kinase cascades and in certain cell lines promote cellular proliferation. The Rho GTPases, including RhoA, Cdc42, and Rac-1, regulate cell proliferation as well as many of the various cell functions mentioned above for small GTPases. It is critical to develop assays for measuring small GTPase activation in order to better understand small GTPase signaling pathways as well as to facilitate discovery of drugs that specifically decrease GTPase activation and ultimately treat several forms of cancer. We propose to develop a new technology that allows for high-throughput and fast analysis of small GTPase activation. An activation specific probe will be fused to glutathione S-transferase (GST) immobilized onto a 96-well microplate to capture activated small GTPases in an extract from cells. Five different activation specific probes from downstream GTPase effecter proteins will be cloned, expressed as GST-fusion proteins, and purified. The binding conditions of small GTPases to the activation specific probes will be developed by using purified recombinant small GTPases. Antibodies raised specifically against small GTPases will identify the bound protein and yield a quantitative result in three hours. Finally, the procedure will be applied to relevant samples such as extracts from cells treated with extracellular mitogenic ligands such as epidermal growth factor (EGF). Such an assay will help to elucidate the molecular mechanisms of proliferative signal transduction pathways and provide means to screen compound libraries to find novel cancer therapy candidates that are direct inhibitors of cancer-causing small GTPase mutants. [unreadable] [unreadable] [unreadable] [unreadable]